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A novel sorting method reveals superior metabolic activity of mononucleated over binucleated tetraploid hepatocytes

Watanabe, Y. V.; Sussman, J. H.; Anami, T.; Hattori, M.; Yamane, S.; Nishikawa, M.; Sakai, Y.; Katsuda, T.

2025-05-07 cell biology
10.1101/2025.05.07.652591 bioRxiv
Show abstract

Hepatocytes display notable ploidy diversity, varying both in the number of genomic copies and in the number of nuclei. Most mouse hepatocytes are tetraploid (4n), which can exist as either mononucleated (1x4n) or binucleated (2x2n) cells. Despite this distinction, these two cell types have traditionally been grouped and studied as a single population. One likely reason for this is that conventional ploidy-sorting methods classify hepatocytes based solely on total DNA content, without distinguishing between mononucleated and binucleated states. In this study, we developed a novel flow cytometry strategy to distinguish and isolate 1x4n and 2x2n hepatocytes. Our approach leverages Hoechst-Area to assess total ploidy and Hoechst-Height to differentiate mononucleated and binucleated hepatocytes. Transcriptome analysis comparing these two populations revealed that 1x4n hepatocytes exhibit a broader and more metabolically active gene expression profile. Importantly, this enhanced metabolic activity was independent of liver zonation, a well-known driver of metabolic heterogeneity in hepatocytes. Our findings uncover a previously underappreciated layer of functional diversity in the liver and provide a new framework for studying the physiological and pathological roles of nuclear configuration in hepatocytes.

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